The Study Of Human Umbilical Cord Wharton’s Jelly Scaffold Loaded Adipose-drived Stem Cells For Construction Of Tissue-engineered Cartilage

Objective: To explore the feasibility of constructing a new type of tissue-engineered cartilage with human umbilical cord wharton’s jelly loaded adipose-drived stem cells.Then evaluate the effect of human umbilical cord wharton’s jelly scaffold loaded adipose stem cells on the repair of rabbit knee joint with full-thickness cartilage defects.Methods:(1)Adipose stem cells were isolated from the subcutaneous fat of New Zealand white rabbits,and the P3 stem cells were identified by flow cytometry.⑵ The induce factor was added to the DMEM(10% FBS,10ng/ml TGF-β1,25ng/ml b FGF,10-7M dexamethasone)which induce cells to the direction of cartilage for two weeks,and the cells were induced by threedimensional induction with the wharton’s jelly scaffold at the final concentration of 10 × 105 / ml to construct tissue engineering cartilage.The distribution and adhesion of the cells in scaffolds were observed by scanning electron microscope(SEM).The levels of extracellular matrix were tested by collagen type Ⅱ immunohistochemical staining,toluidine blue and safranine staining.The viability and proliferation of the cells were evaluated by laser scanning confocal microscopy after FDA-PI staining and Hoechst 33258 staining of frozen section.⑶ A diameter of 4mm,2mm deep full-thickness cartilage defects was created in the rabbit knee cartilage,and in this experiment we set the blank group,simple scaffold material group and cellscaffold complex group.The histological staining,glycosaminoglycan,total collagen content and biomechanical analysis of defect repair area at 24 weeks postoperatively to evaluate the effect of repair.Results:(1)Flow cytometry showed that CD73,CD90,CD105 were positive,CD34,CD45 and HLA-DR were negative,suggesting that the cells were adipose derived stem cells.(2)Histological staining indicated that the cells secret a large number of extracellular matrix.It was observed by SEM that a large number of cells were adhered to the pores of scaffold and the cellular morphology were round or oval.After PDA-PI staining and Hoechst 33258 staining,we can see that the cells were adhered to the scaffold,and most of them were living cells,the cell survival rate was as high as 90%.(3)24 weeks after the surgery,defective area of cell-scaffold complex group was mostly repaired,and the defect was filled with cartilage tissue,repair area has a smooth surface.Histological staining showed a typical hyaline cartilage like structure,the level of GAG,total collagen quantitative can up to 90% of the normal tissue,biomechanical measurements suggest repair area was similar to normal cartilage.In the scaffold group,most of the defective areas were repaired,but the repair area was mostly fibrous cartilage,with only a few hyaline cartilage like structures.The blank group failed to repair articular cartilage defects,fibrous tissue partially filled with defects,and the surface covered with lamellar fibrous tissue.Conclusion: Adipose stem cells are feasible as cartilage tissue engineering seed cells.Human umbilical cord wharton’s jelly scaffold can be used as a good scaffold for cartilage tissue engineering.Experimental tissue cartilage can effectively repair articular cartilage defects in the long run.